Studies in prebiotic synthesis

J. Mol. Biol. (1970) 47, 531443

Studies in Prebiotic Synthesis V.t Synthesis and Photoanomerization

of Pyrimidine Nucleosides

ROBERT A. SANCHEZ AND LESLIE E. ORGEL

The Salk Institute San Diego, Calif. 92112, U.X.A. (Received 23 June 1969, and in revised form 15 September 1969) n-Ribose-5-phosphate, D-ribose and D-arabinose react with cyanamide in aqueous solutions to produce aminooxazoline derivatives. These in turn react with cyanoacetylene to yield, respectively, a-5’-cytidylic acid, cr-cytidine and j3arabinosylcytosine. Anomerization and epimerization of these and related nucleosides and nucleotides occur when they are irradiated in aqueous solution with an unfiltered 253 rnp light source. The possible relevance of these reactions to prebiological chemistry and their application to the synthesis of nucleosides are discussed.

1. Introduction of cytosine and uracil from cyanoacetylene under potentially prebiotic conditions has been reported (Ferris, Sanchez & Orgel, 1968). We describe here a related synthesis of cc-ribosylpyrimidines and ,&arabinosylpyrimidines, and some novel photoisomerization reactions of these and other pyrimidine nucleosides. These reactions may be relevant to the prebiological synthesis of pyrimidine nucleosides and may also have some role in the photochemistry of nucleic acids. A synthesis

2. Materials and Methods (a) General Cytidine and deoxycytidine were A-grade chemicals from Calbioohem. Arabinosylcytosine was purchased from Pierce Chemical Co., and bacterial alkaline phosphatase from Worthington. Cyanoacetylene was prepared by the dehydration of propiolamide with PsO, (Ferris et al., 1968). All other reagents were commercial products of the best available quality. Descending paper chromatography and electrophoresis were done on Whatman 3MM paper. The following systems were most frequently used: A, iso-propyl alcohol-cone. ammonia-water (7 : 1: 2 by vol.) ; B, n-propyl aloohol-conc. ammonia-water (11: 2 : 7 by vol.); C, rr-butyl alcohol-5 N-acetic acid (2: 1 v/v); D, water-saturated n-butyl alcohol; E, tetrahydrofuran-88% formic acid-water (3: 1: 1 by vol.); F, electrophoresis, 4 kv in 0.05 N-formate, pH 2.6; G, electrophoresis, 4 kv in 0.03 N-phosphate, pH 7.1; H, electrophoresis, 1 kv in 0.05 N-borate, pH 9.2. Solutions in quartz tubes were irradiated in a Rayonet photochemical reactor at approx. 35%; the manufacturers claim an output of 12800 microwatts/cma at 2537 8. Ultraviolet spectra were recorded on a Unicam SPSOO spectrophotometer, and optical rotatory dispersion spectra on a Jasco Spectropolarimeter model UV/ORD-5. Column chromatographic separations were monitored spectrophotometrically with a Gilson fraction collector. t Paper IV in this series is Ferris, Sanchez & Orgel, 1968. 631

532

R. A. SANCHEZ

AND

L. E. ORGEL

(b) Prqoarative scale synthesis from D-riboss&phosphate (i) a-5’-Cytidylic acid Solutions of o-ribose-B-phosphate (3 m-moles in 3 ml. of 1 N-NH,) and cyanamide (6 m-moles in 27 ml. of water) were mixed and heated on a steam bath for 15 min, then 9 m-moles of cyanoacetylene were added and heating was continued for 15 hr. The clear reddish solution was concentrated and chromatographed on a column of cellulose using solvent B. The combined cytidylio acid fractions (located by their ultraviolet spectra) were concentrated and applied to a cohunn of Dowex 2 (X&ZOO (formate)) and eluted first with water and then with 0.1 M-HCOOH. The combined cytidylic acid fractions (39% yield, estimated from the ultraviolet absorbance) were applied in a small volume to a column of Amberlite CG50 and eluted with water. A single major band of cytidylic acid was obtained. Freeze-drying yielded 300 mg (28%) of product as a cream-colored powder: analysis talc. for C9H14N308P * NHe * Hz0 : C, 30.20 ; H, 5.36 ; N, 15.62 ; P, 8.65. Found : C, 3062; H, 5.58; N, 1551; P, 8.66. In a similar preparation to which /3-5’-[2-f4C]cytidylic acid had been added, the radioactive marker coincided with the synthetio product throughout the separations. (ii) a-Cytidine 50 mg of the above-synthesized a-5’-cytidylic aaid in 6 ml. of 0.1 M-Tris buffer at pH 8.2 was incubated overnight at 3’7°C with alkaline phosphatase (12 units, Worthington BAPF), then concentrated and applied to a column of Dowex 1(X10-200 (OH-)). Elution with 300/e CH,OH (Dekker, 1965) yielded a major fraction of a-oytidine (90% yield) and a minor peak (0.4% yield) whose retention volume corresponded to that of fi-oytidine. (c) Structure proof

of E-5’-cytidylic

acid and cwytidine

(i) Ch?+omtography Synthetic a-5’-cytidylic acid and authentic /3-5’-cytidylic acid were almost undistinguishable in all our chromatographic systems (A to H included) and by ion-exchange chromatography. The best separation was obtained in system E in which the mobility of E-5’-cytidylic acid relative to the /3-anomer was 1.15. cc-Cytidine and /3-cytidine were also difficult to separate in the usual systems. However, a complete separation could be effected by column chromatography over Dowex 1 (OH-) with CHsOH-water mixtures (Dekker, 1965) (ii) Uttraviolet spectra The ultraviolet spectra of the ences. Similar observations are 2’- and 3’-cytidylic acids (Table The uhraviolet spectra of the ences.

anomeric 5’-cytidylic acids show small but distinct differdescribed by Gassen & Witzel (1965) for the anomeric 1). anomerio cytidines also show these characteristic differ-

(iii) Optica rotatory dispersion spectra The optical rotatory dispersion spectral data for a-5’-cytidylic acid are compared in Table 2 with the values reported by Nishimura, Shimizu & Iwai (1968). Data for the /3-anomers are also included. Similar optical rotatory dispersion spectra are obtained from the corresponding cytidine anomers. (iv) Periodate czeavage-borohydrtie reduction The anomeric configuration of a-cytidine was further confirmed using a technique described by Davoll, Lythgoe & Todd (1946) and Wright, Tener & Khorana (1958). Oxidation with sodium periodate followed by reduction with sodium borohydride yields a glycolether in which only the C1’ position remains asymmetric. The compounds obtained in this manner from cc- and 6-cytidine had identical chromatographic mobilities and ultraviolet spectra, and mirror-image optical rotatory dispersion spectra.

acid

acid

acid

acid acid

B-2’-Cytidylic

x-3’-Cytidylic

/?-3’-Cytidylic

a-5’-Cytidylic p-5’-Cytidylio

280 279.5

278.5

280

277

278.5

h max (mp)

The data for the 2’- and 3’-isomers the present work.

acid

e2’Xytidylic

Compound

TABLE

1

8.3 7,6

6.8

6.4

5.9

6.6

A max/min

are from Gassen & Witzel

240.5 240.5

240.5

240.5

241

241

pHlto3 h min (II+)

249 250

251

250

252

251

pH7to9 X mm (mp)

are from

1.63 1.44

1.36

1.53

1.24

1.40

A max/min

(1965). The data for the 5’-isomers

271.5 271

270

271.5

269.5

270.5

A max (my)

Ultraviolet absorption spectra of the anomeric cytidylic acids

acid$

acid?

acid3

a-5’Xytidylic

/l-5’-Cytidylio

/3-5’Zytidylic

271

271

271.5 288

290

288

289

+

8.7

6.7 273

275

271

+

273

-15-o

bv4

Crossover

- 12.3

(mr) 271.5

First extreme

(mp) ([MI x lo-*)

x max

250 to 235

250 to 235

250 to 236

250 to 235

(w.4 X

10-3)

acids

- 12.4

- 11.2

t-15.0

+ 15.8

([iv]

Second extreme

t From the data of Nishimura et al. (1968). Spectra measured in water at pH 7. $ From the present work. Spectra measured in 1 y0 phosphate buffer at pH 7.0.

acid?

a-5’-Cytidylio

Compound

Optical rotatory dispersion spectral data of the anomeric 5’.cytidylk

TABLE 2

Amplitude

+21*1

+17*9

- 30.0

-28.1

([Ml X lo-?

SYNTHESIS

OF

PYRIMIDINE

535

NUCLEOSIDES

(d) Effects of certain variables in the synthesis of u-5’-cytidylic acid (i) Comparison of conden&ng agents A series of mod&d reactions, similar to the one described in section (b) (i) but on a smaller scale, was carried out. Each reaction mixture was then analyzed by paper chromatography (system B) to determine the yields of m-5’-cytidylic acid. n-Ribose-&phosphate, ammonia, cyanamide and cyanoacetylene in the pH range of 7 to 10 gave yields between 20 and 40%. The replacement of cyanamide by an equivalent concentration of cyanogen gave yields of 2 to 7 o/o; cyanate, dimethylcyanamide and oyanogen bromide gave little or no cytidylio acid. In the absence of ammonia (pH adjusted to 8 to 9 with NaOH) the yields of cytidylic acid were 20% from cyanamide and 0.2% from cyanogen. (ii) Variability of the a/p anomer product ratio Purified cytidylic acid from each of the experiments in section (d) (i) was dephosphorylated enzymically and chromatographed over Dowex 1 (OH-). Each product was predominantly the a-anomer; none contained more than 1 to 2% of the 8-anomer. In another experiment, a synthesis similar to the one described in section (b)(i) was carried out. Prior to adding oyanoacetylene, portions of the reaction mixture were irradiated at 253 mp, heated with sodium cyanide or with sodium iodide. The purified product from each of these reactions contained less than 1% of the j3-anomer. (iii) Effect of initial concentratiolzs on product yields Solutions containing D-ribose-&phosphate, ammonia and cyanamide in molar ratios of 1: 2 : 2 were warmed at 60°C for 1 hr, then cyanoacetylene was added (molar ratio 3). The solutions were heated at 6O’C for 1 hr and then at 100°C for 1 hr. All reactions were kept at a pH of about 9 to 10 with additions of NaOH. Samples corresponding to 2 pmoles of n-ribose-5-phosphate were concentrated and analyzed by descending paper chromatography in system B. The ultraviolet-absorbing spots of cytidylic acid, clearly visible in each case, were eluted and the yields estimated from the ultraviolet spectra. The results are summarized in Table 3.

TABLE 3 In&hence

oj ribose-5-phsphate

concentrations

on cytidylic

Ribose&phosphate (mole/l.)

a-6’-Cytidylic acid yield (%I

0.10

20.0 18.9 13.4 3.6 2.8

0.030 0.010 0*0030 0~0010

acid yields

(e) Reactions of other sugars (i) D-Ribose The reaction of n-ribose with ammonia, cyanamide and oyano-acetylene (under the conditions used for the synthesis of a-6’-cytidylic acid; sections (b) and (d) ) produces acytidine in yields of 10 to 20%. If cyanogen is used in place of cyanamide then yields of 1 to 3% are obtained. The a-cytidine is identical in all respects (ultraviolet and optical rotatory dispersion spectra, paper and column chromatographic properties) with that obtained by enzymic dephosphorylation of the a-5’.oytidylic acid obtained from n-ribose5-phosphate (section (b) (ii) ). A second, faster moving product (systems A to D) is formed in yields roughly equal to those of a-cytidine, and is thought to be I-a-D-ribopyranosyl oytosine. SK

636

R. A. SANCHEZ

AND

L. E. ORCEL

(ii) D-Ar&nose Under conditions identical to those used in the reactions of n-ribose, n-arabinose produced p-arabinosylcytosine in yields of about 10%. The product was identical to an authentic standard as judged by chrometographic mobility (systems A, C, D and F) and ultraviolet spectra. The reactions of D-arabinose are described in more detail in sections (f) and (g). (iii) 2-Deoxy-D-ribose One ml. of a solution of 2-deoxy-n-ribose (O-10 M), ammonia and cyanamide (0.20 M each) heated at 60°C for 1 hr. Cyanoacetylene was added (O-30 M) and the solution heated for a further 10 min and then at 100°C for 1 hr. Paper chromatography revealed the presence of many ultraviolet-absorbing products. Tritiated fi-deoxycytidine was added (to about 4 x 10m6 M) and chromatographio separations of the eluted radioactive areas were carried out successively in systems A, C, D and F. The final product was chromatographitally homogeneous and the ultraviolet absorbance indicated a yield of 0.6%. However, the ultraviolet and optical rotatory dispersion spectra both indicated that the product was the cr-anomer (cf. Table 6) and contained no more than about 2 to 3% of the /3-anomer. In exactly parallel reactions analyzed in the same way, D-ribose gave a 14% yield of a-cytidine and n-arabinose gave an 8.4% yield of /3-arabinosylcytosine. was

intermediates (f) Synthf&s of amino-oxazoline (i) %Amino-cc-o-ribofurano [I’, 2’: 4, 51 2-oxazoline (from D-ribose) A solution of n-ribose (O-10 mole) and cyanamide (O-20 mole) in 100 ml. of 1 N-NH, was heated at 60°C for 1 hr, then refrigerated overnight at 4°C. A first crop of crystals was removed, then the filtrate was evaporated and stirred with methanol to yield a second crop. The combined yield was 15.1 g (87%). Recrystallization from water gave a pure white, finely grannlar solid, m.p. 195°C (decomp.): analysis talc. for C6H10N204: C, 41.40; H, 5.79; N, 16.08; 0, 36.73. Found: C, 41.54; H, 6.15; N, 15.99; 0, 36.74. (ii) 2-Amigo-B-o-arabinoficra~o [I’, 2’: 4, 51 2-oxasoline (from D-arabinose) A solution of n-arabinose (0.10 mole) and cyanamide (0.20 mole) in 30 ml. of 1 N-N& w&s heated at 60°C for 30 min, then chilled in an ice-salt bath and seeded with crystals of the product. The yield of white solid, m.p. 167 to 169°C (decomp.), was 7.6 g (44%). This product is more water-soluble than that obtained from n-ribose. Higher yields are obtained (60 to 80%) by conducting the reactions in aqueous-methanolic solutions. Recrystallization from CHaOH-water yields a pure white micro-crystalline powder, m.p. 1750 to 175.3% (decomp.): analysis talc. for CsHIoNzO,: C, 41.40; H, 5.79; N, 16.08; 0, 36.73. Found: C, 40.97; H, 6.70; N, 16.08; 0, 37.14. The structure of these amino-oxazolines follow from the methods of synthesis, the elemental analyses and the structures of the reaction products (section (g)). (g) Reactions of the amino-oxazoline (i) With cyanoacetylene in water The amino-oxazolines (section (f), 0.10 M) reacted with cyanoacetylene (0.1 to 0.3 M) in aqueous, unbuffered solutions (pH 8 to 9) at 60 to lOO”C, yielding in each case the same products as those obtained from reactions of the parent sugar with cyanamide and oyanoacetylene (section (e) ) . Solutions of the amino-oxazolines and cyanoacetylene (each 0.10 M, pH 8.5) were heated at 60°C and the progress of the reactions was followed spectrophotometrically. The typical cytidine-like spectra appeared cleanly in each case, with half-lives of about 5 min at 60°C. The final yields were 64% from aminoribofurano-oxazoline and 58% from aminoarabinofurano-oxazoline. (ii) W6th cyanoaoetylem in. N,N-dim&hylucetuetam/ide The reaction of aminoarabinofurano-oxaeoline with cyanoaoetylene in N,N-dimethylacetamide was carried out a number of times and studied in some detail, with the following results :

SYNTHESIS

OF PYRIMIDINE

NUCLEOSIDES

637

Equimolar solutions of &mino&r~binofurcmo-oxazoline and cyanoacetylene (0.10 to 1.0 in N,iV-dimethylscetamide react rapidly at room temperature (within about 30 min) to produce a clear yellow solution in which no further change occurs upon heating. An infrared spectrum of the solution (thin &) shows a C zz N stretching frequency at 2214 (acrylonitrile cm-l which we attribute to the cyanovinyl adduct of the amino-oxazoline in N,N-dimethylacetamide absorbs Bt 2224 cm-l). The ultraviolet spectrum of a sample diluted into water (pH 6 to 7) shows an absorption with X max 258 mp (c max 16000, assuming 100% reaction). This spectrum shifts with a half-life of 3 min to a new absorption of h max 263 rnp which we attribute to 02,2’-cyclooytidine. Chromatography (systems C and D) of concentrated aqueous mixtures of the N,N-dimethylecetamide reaction solution show 0,2’-cyclocytidine to be the initial product of hydrolysis. Subsequently, increasing amounts of b-cytosine arabinoside are formed. The ultraviolet spectra of the eluted Oa,2’-cyclocytidine and of a sample prepsred by the procedure of Walwick, Roberts & Dekker (1959) were identical in all respects. Complete aqueous hydrolysis (e.g. 5 mm in 1 N-NH, at SO’C) of the N,N-dimethylacetamide reaction mixture produces ,S-arabinosylcytosine in yields of about 900/& measured spectrophotometrically. In two separate preparations the N,N-dimethylacetamide solutions were hydrolyzed in warm 1 N-NH,, evaporated to dryness, treated with methanolic HCI and worked up to give crystalline /3-arabinosyleytosine hydrochloride in yields of 82% and 84%. The melting points, chromatographic mobilities (systems A to H), ultraviolet, optical rotatory dispersion and infrared spectra of these products were identical to those of authentic samples of /3-arabinosylcytosine hydrochloride. M)

(iii) With methyl propiokzte A solution of sminoribofurano-oxazoline (9.8 m-moles) and methyl propiolate (20 mmoles) in 10 ml. of 1 N-NH3 was heated at 60°C for 15 min and then at 100°C for 30 mm. Workup of the mixture yielded 0.86 g (36% yield) of the a-anomer of 02,2’-oyclouridine, m.p. 223 to 225°C: analysis talc. for C,HI,Ns05: C, 47.80; H, 4.46; N, 12.41; 0, 35.35. Found: C, 48.02; H, 4.65; N, 11.93; 0, 36.20. Acid-catalyzed hydrolysis of this compound produced a-uridine (identical to the nitrous acid deamination product from cc-oytidine) in good yields. In a similar fashion aminoarabinofurano-oxazoline (9.8 m-moles) and methyl propiolate (12 m-moles) yielded 0.42 g (18% yield) of Oa,2’-cyclouridine, m.p. 247 to 249°C : analysis talc. for CgHIoN,OS: C, 47.80; H, 4.46; N, 12.41; 0, 35.36. Found: C, 47.76; H, 4.69; N, 11.92; 0, 35.60. The ultraviolet spectrum of this compound is the same as that reported by Letters & Michelson (1961) for 02,2’-cyclouridine. Acid-catalyzed hydrolysis produced fi-arabinosylumcil in good yields. (h) P~otoch.em&ry (i) Iwadiation of nzucleosides Irradiated solutions were concentrated and subjected to descending chromatography in system A or electrophoresis (system F). These systems separated the combined “cytosine nucleosides” from various other photo-products such as uridines, free bases, stable hydrates, eto. The eluted cytosine nuoleosides were then concentrated and applied in small volumes to columns of Dowex 1 (OH-);usually the Bio-Rad product AGl-X2-400 (Dekker, 1965). The elution characteristics of various nucleosides are summarized in Table 4. The fractions from each produet were pooled and the ultraviolet spectra measured. Yields were calculated by comparing the absorbanoe values with those of unirradiated controls that had been carried through the same separation procedures, The results of several typical reactions are summarized in Table 5. (ii) Irradiation of nuoleotidea After irradiation the solutions were concentrated and chromatographed in system A or B. The combined nucleotides (free from all photodephosphorylated products) were eluted and dephosphorylated with alkaline phosphatsse. Subsequent steps in the analysis were the same as those described in (i).

12. A. SANCHEZ’

53.8

AND

L. E. ORCEL

TABLE 4 Relative

retention volwnes on Dowex

Compound

1 (OH)

30% or 60% CH,OHT

Water

0.7 1.0 1.3 1.8 <0*3 so.3

-

a-Cytidine ,&Cytidine a-Arabinosyloytosine &Arabinosylcytosine u-2’-Deoxycytidine fl-2’-Deoxycytidine t Relative retention volumes in water compared mately 36, 4 and 1, respectively.

0.7 1.0

to 30% CH,OH and 60% CH,OH

are approxi-

Irradiation in the presence of added substances (0.01 N-N~~HPO~, NH,, N&N or MgCls, all adjusted to pH of about 8) resulted in changed rates or yields, but did not signihcantly change the ratios of the resulting u- and fi-cytidines. Purging with nitrogen had no significant effect on the reaction. (iii)

Ider#ication

of photoproducts

a- and ,%Cytidine and ,%arabinosylcytosine were identified by comparison with authentic samples. Their column retention volumes, relative mobilities on paper chromatography in at least two systems, ultraviolet and optical rotatory dispersion spectra were identical. The ultraviolet and optical rotatory dispersion spectral data are summarized in Table 6. A bioassay using Escherichia coli BlO (pyrimidine-requiring strain) further conhrmed the identity of photosynthesized /?-cytidine (Table 7). The sugar moiety of photosynthesized /3-arabinosylcytosine was further proved by chemical degradation of the nucleoside (Cohn & Doherty, 1956) followed by chromatogmphic identification of wrabinose in system C. cc-2’-Deoxycytidine has not been reported in the literature. The properties of x-2’deoxycytidine and j3-2’-deoxyoytidine are related in very much the same way as those of a-cytidine and /3-cytidine (Tables 4 and 6). On this basis we feel confident of the structure assignment. The identification of a-arabinosylcytosine is tentative. Its ultraviolet spectra in acid and in base are almost identical to those of the @momer and both compounds fail to complex with borate as evidenced by lack of mobility on electrophoresis at pH 9.0 in borate buffer. This indicates that cis OH groups are absent. (iv) Iwadiation

of other bme.9, etc.

5 x 10e4 M-solutions of commercial edenylic acid and deoxyadenylic acid in quartz tubes of 27 mm diameter were irradiated 4 days. The nucleotides were recovered in greater than 95% yield. Analysis as described in section (c) indicated less than 0.1 oh anomerization. 100 mg of calf thymus DNA (Sigma, type I) in 200 ml. of buffer (O-01 M-citrate, 0.001 M each of Tris and EDTA, pH 8.2, nitrogen purged) was irradi&ed for 5 hr. The resulting solution was dialyzed, hydrolyzed with 0.1 an-HsS04 at 1OO’C for 4 hr and dephosphorylated with alkaline phosphatase. Deoxycytidine was separated and analyzed by procedures similar to those described in section (c). Chromatography on Dowex 1 g&ve a peak corresponding to a-2’-deoxycytidine, in a yield of about 1% relative to the recovered p-2’-deoxycytidine. Denatured DNA gave much less a-2’-deoxycytidine; an unirradiated control gave none. The total amounts of 2’-deoxycytidine that were recovered in these experiments were in the approxim&te ratio of 6 : 9 : 10, respectively.

27 27 27 27

2x10-4 2x IO-4 2x10-3 2x 10-s

p-Arabinosylcytosine

fi-Cytidine b-2’-Deoxycytidine

acid

acid

a-5’Xytidilic

,6-B’-Cytidylic

t Present in small amounts but not measured. $ Yield uncertain due to overlap from the &cytidine 8 These are a- and ,%deoxycytidine.

1 1

62 2

peak.

16

IlO

410 6

128 32

16

na

2

37

3

-

3

1

7 7

no STes yes

2 2

t

4 62

5

nucleosides a-Cytosine arabinoside

58

4 4

6

cc-Cytidine

no

n0

at a pH of 8.

8

2x 10-s

/?-Cytidme

were in 0.01 M-NH~HCO~

8

All solutions

18

2x IO-3 2x 10-s

Irradiation tima (hr)

Product cytosine ,&Cytidine

Nitrogen flush

Tube diameter e4

Moles/l.

a-Cytidine

Compound

reactions of cytosine nucleosides and nucleotides

Photoisomerization

TABLE 5

1

3

5

6

86

t

(%) /3-Cytosine arabinoside

249.0 250.0 260.0 248.0

2715 271.5 2705 270.5 270.5

authentic

fi-Cytidine

authentic

a-Cytidine

/3-2’-Deoxycytidine

authentic

a-Cytidine

fi-Cytidine

j-Cytidine

a-2’-Deoxycytidine

j3-2’-Deoxycytidine

/3-Arabinosylcytosine

b-Arabinosylcytosine

249.5

271.0

authentic

i Ultraviolet reported.

and optical

rotatory

dispersion

data of the arabinosides

buffer at pH 7-O. All concentrations

249.5

271.0

a- or flcytidinet

Spectra measured in 1% phosphate

260.0

270.5

249.0

h mm (mp)

a-Cytidine

h max (mp)

Source

Compound

Ultraviolet

287

x

235-250

272

230-250

230-250

240-250

within

experimental

lo-“)

- 17.2

- 18.9

-6.6

+ 15.1

- 13.0

-11.5

+ 18.0

+17,1

X

Averages

cells). error.

about l-5 in lo-mm

271

271

272

2406250

235-250

272 272

240-250

272

272

([M]

Second extreme

(w) 240-250

dispersion

Crossover

rotary

10m4 M (absorbance

+ 18.1

+ 16.2

+6*6

- 12.5

+8,3

+8*1

- 15.9

- 18.3

@I X 10-3)

from both anomers were identical

were about 1.7

1.34

287

287

1.40 1.43

287

288

288

288

288

h-4

162

1.36 1.36

1.65

164

A max/min

First extreme

Optical

Ultraviolet and optical rotatory &person data of cytosine nucleosides

TABLET

are

SYNTHESIS

OF

PYRIMIDINE TABLE

NUCLEOSIDES

541

7

Bioassay of u- and p-cytidines with E. coli BlO Source

Added nucleoside None /3-Cytidine p-Cytidine a-Cytidine

O.D.

(600 mp) 0.003 0.300 0.270 0.002

authentic from irradiated a-oyticline authentic, u&radiated

In a mineral medium containing histidine, thiamine and glucose. Nuoleoside were 10 pg/ml. Incubations were for 12 hr at 37”C, after which time absorbance recorded as a measure of turbidity resulting from cell growth.

concentrations at 600 mp was

(i) Hydrolyses Solutions of a-B’oytidylic acid and ,3-5’eytidylic acid (each about 0.02 M) were prepared in 2% potassium phosph&+sodium borate buffer solutions (pHydrion) and adjusted to a pH of 8.50. Samples wore sealed into small glass tubes and placed in a steam bath at 100°C. Analyses were made periodically. All of the ultraviolet-absorbing hydrolysis products were separated by chromatography in systems C and F, and yields were estimated from the ultraviolet absorbance of the eluted spots. The results are illustrated in Fig. 1. The initial hydrolysis rates at 60°C were about 1.5% that of the rates at 100°C. Under similar conditions but at a nH of 7.5, B-evtidine has a half-life of 10 days at 100°C and 780 days at 60°C. Uridine is formed in good yyelds.

P 45’60-

\.

s

(al Fro. 1. Hydrolysis of anomeric phate buffers at pH 8.50.

Time (weeks 1

5’-cytidylic

(b)

aoicls at 100°C. Solutions

were 0.02 M in 2% phos-

3. Results D-Ribose-5-phosphate reacts with solution to give cc-5’-cytidylic acid in

cyanamide and cyanoacetylene in aqueous up to 40% yield. D-Ribose and D-arabinose yield a-cytidine and &arabinosylcytosine, respectively. The products contain very little of the anomeric nucleosides or nucleotides. The reaction with %deoxy-D-ribose is very much less eflicient, yielding less than 1% of a-deoxycytidine.

R.

542

A.

SANCHEZ

AND

L.

E.

ORGEL

Isolable intermediates, amino-oxazolines, are involved in these syntheses. The amino-oxazolines react with cyanoacetylene in water or in organic solvents to give adducts which ring close to cyclooytidines. These latter finally hydrolyze in water to nucleosides of fixed anomerio configuration (Pig. 2). ,%Arabinosylcytosine

Ddrabinose

OH

OH NH2CN I

NH H20

(phas) HOCH, 0 OH D-

OH

OH

@-@ibose (D-Ribose-5-phosphate)

G-Cytidine k&Cytidylic

FIO. 2. Reaction

of D-ribose and n-arabinose

with cyanamide

acid)

and oymometylene.

Similar reactions occur when cyanamide is replaced by cyanogen and ammonia. Uracil derivatives are obtained directly if oyanoacetylene is replaced by propiolio ester. Photoanomerization and photoepimerization occur when cytidine (a or p) or deoxycytidine is irradiated in aqueous solutions with an unfiltered 253 mp light source. Yields are given in Table 5. Hydrolysis of a-oytidylic acid gives small amounts of cr.aridylic acid and cc-cytidine,

SYNTHESIS

OF

PYRIMIDINE

NUCLEOSIDES

543

but the yields are smaller than those of corresponding compounds obtained from /3-cytidylio acid under the same conditions (Fig. 1).

4. Discussion The dark reactions which we have described lead to the formation of derivatives of cc-cytidine and ,%arabinosylcytosine, but not to derivatives of the important nucleic acid component /I-cytidine. The photochemical isomerization of a-cytidine or flarabinosylcytosine does give /I-cytidine but in relatively low yields (about 5%). Thus while we can propose a “prebiotic” route to oytidine and uridine, it is not very efficient. Nonetheless, it is the most plausible prebiotic route to any of the natural nucleosides that has been reported. Pyrimidine nucleotides in contemporary ribonucleic acids are j%ribosides, and so we can only speculate if compounds such as u-cytidine and &arabinosylcytosine might have contributed to the prebiotic evolution of nucleic acids. The photochemical isomerization reactions, although relatively inefficient, would have provided small amounts of /3-ribosides. The eflicient hydrolysis of /3qtidylic acid to /3-uridylio acid shows that a synthesis of the former compound would automatically lead to a supply of the latter. The amino-oxazolines are novel organic intermediates. Preliminary experiments suggest that they are formed by many sugars and will be useful for the synthesis of a wide variety of nucleosides and nucleotides in addition to those discussed in this paper. This work was supported by grant GB5303 of the National Science Foundation. We acknowledge the extensive and very capable assistance of Mr Robert Holland and Mr Robert

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